Localization of human recombinant adenoviral vectors to the mitochondria following transduction of human cell lines.

Recently, mitochondria have been shown to have a vital role in the innate immune response to viral infection. In response, viruses such as adenovirus, have developed mechanisms to alter mitochondrial function through direct or indirect interaction with the mitochondria. The interaction of human recombinant adenoviral vectors directly with human mitochondria has not previously been shown. We demonstrate that human recombinant adenoviral vectors co-localize to mitochondria. We show that the adenoviral vectors are present within the membranes of the mitochondria and that they cause ultrastructural changes to the cristae. Further, we show that the adenoviral genome is also present in intact mitochondria. We have posited that the interaction between the adenovirus and the mitochondria may act to inhibit mitochondrial function. We have also posited that the transport of the adenoviral genome to the mitochondria may allow the future use of this vector as a tool for gene therapy of mitochondrial diseases. Keywords: mitochondria; human recombinant adenovirus; gene therapy; viral vectors; mitochondrial DNA; electron microscopy.

Identifying early events of gene expression in breast cancer with systems biology phylogenetics.

Advanced omics technologies such as deep sequencing and spectral karyotyping are revealing more of cancer heterogeneity at the genetic, genomic, gene expression, epigenetic, proteomic, and metabolomic levels. With this increasing body of emerging data, the task of data analysis becomes critical for mining and modeling to better understand the relevant underlying biological processes. However, the multiple levels of heterogeneity evident within and among populations, healthy and diseased, complicate the mining and interpretation of biological data, especially when dealing with hundreds to tens of thousands of variables. Heterogeneity occurs in many diseases, such as cancers, autism, macular degeneration, and others. In cancer, heterogeneity has hampered the search for validated biomarkers for early detection, and it has complicated the task of finding clonal (driver) and nonclonal (nonexpanded or passenger) aberrations. We show that subtyping of cancer (classification of specimens) should be an a priori step to the identification of early events of cancers. Studying early events in oncogenesis can be done on histologically normal tissues from diseased individuals (HNTDI), since they most likely have been exposed to the same mutagenic insults that caused the cancer in their neighboring tissues. Polarity assessment of HNTDI data variables by using healthy specimens as outgroup(s), followed by the application of parsimony phylogenetic analysis, produces a hierarchical classification of specimens that reveals the early events of the disease ontogeny within its subtypes as shared derived changes (abnormal changes) or synapomorphies in phylogenetic terminology.

Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and neurodegenerative disease in mice.

In mammalian cells, regulation of the expression of proteins involved in iron metabolism is achieved through interactions of iron-sensing proteins known as iron regulatory proteins (IRPs), with transcripts that contain RNA stem-loop structures referred to as iron responsive elements (IREs). Two distinct but highly homologous proteins, IRP1 and IRP2, bind IREs with high affinity when cells are depleted of iron, inhibiting translation of some transcripts, such as ferritin, or turnover of others, such as the transferrin receptor (TFRC). IRPs sense cytosolic iron levels and modify expression of proteins involved in iron uptake, export and sequestration according to the needs of individual cells. Here we generate mice with a targeted disruption of the gene encoding Irp2 (Ireb2). These mutant mice misregulate iron metabolism in the intestinal mucosa and the central nervous system. In adulthood, Ireb2(-/-) mice develop a movement disorder characterized by ataxia, bradykinesia and tremor. Significant accumulations of iron in white matter tracts and nuclei throughout the brain precede the onset of neurodegeneration and movement disorder symptoms by many months. Ferric iron accumulates in the cytosol of neurons and oligodendrocytes in distinctive regions of the brain. Abnormal accumulations of ferritin colocalize with iron accumulations in populations of neurons that degenerate, and iron-laden oligodendrocytes accumulate ubiquitin-positive inclusions. Thus, misregulation of iron metabolism leads to neurodegenerative disease in Ireb2(-/-) mice and may contribute to the pathogenesis of comparable human neurodegenerative diseases.

Use of a novel virus detection assay to identify coronavirus HKU1 in the lungs of a hematopoietic stem cell transplant recipient with fatal pneumonia.

A 38-year-old female patient with systemic lupus erythematosus presented with pulmonary infiltrates and hypoxemia for several months following immunodepleting autologous hematopoietic stem cell transplantation. She was treated for influenza, which was isolated repeatedly from oropharynx and bronchoalveolar lavage (BAL) fluids, and later empirically for lupus pneumonitis, but died 6 months after transplant. Autopsy findings failed to show influenza in the lungs or lupus pneumonitis. A novel generic polymerase chain reaction (PCR)-based assay using degenerate primers identified human coronavirus (CoV) HKU1 RNA in BAL fluid at autopsy. CoV was confirmed by virus-specific PCRs of lung tissue at autopsy. Electron microscopy showed viral particles consistent with CoV HKU1 in lung tissue both at autopsy and from a previous biopsy. Although human CoV HKU1 infection is not usually severe, in highly immunocompromised patients, it can be associated with fatal pneumonia.

Death by edible mushroom: first report of Volvariella volvacea as an etiologic agent of invasive disease in a patient following double umbilical cord blood transplantation.

We describe a case of invasive fungal infection caused by Volvariella volvacea following double umbilical cord blood transplantation (UCBT). Although infections caused by several mushroom species have been documented, we believe this to be the first published report of invasive infection with Volvariella volvacea, an edible mushroom belonging to Agaricales.

Immunohistochemical and ultrastructural localization of leptin and leptin receptor in human white adipose tissue and differentiating human adipose cells in primary culture.

Leptin is mainly produced in white adipose tissue and acts both at distant sites and locally at the tissue from which it originates. The cellular and subcellular localization of leptin and its receptor (Ob-receptor [Ob-R]) and their relationship to various stages of fat cell maturation have not been characterized as yet. Therefore, we analyzed leptin and Ob-R by using reverse transcriptase-polymerase chain reaction, immunohistochemistry, and ultrastructural immunogold labeling in human white adipose tissue and in human adipocyte cell cultures at early and late stages of differentiation. Both leptin and its receptor were present in mature unilocular fat cells. The thin cytoplasmic rim of the adipocytes exhibited the strongest expression of both leptin and Ob-R. At early stages of differentiating human adipocytes, leptin was mainly expressed in multilocular preadipocytes, whereas the Ob-R was found predominantly on fibroblast-like cells. Other cellular components of human white adipose tissue were characterized by anti-CD31 for endothelial cells, anti-CD68 for macrophages, and antibodies specifically labeling B-cells and T-cells. In addition to fat cells, endothelial cells were immunopositive for the full-length leptin receptor. On the ultrastructural level, leptin was mainly found attached to cellular membranes and in small alveolate vesicle-like structures in the cytoplasm of adipocytes. Leptin was also present on the cell membranes of endothelial cells and macrophages. We conclude that the expression of the Ob-R in human white adipose tissue is not restricted to adipocytes but is present in resident endothelial and immune cells. Ultrastructural localization studies revealed an association of leptin with cell membranes and small vesicles. The cellular and subcellular distribution of leptin and its receptor suggests an important autocrine and paracrine role for leptin in human adipose tissue.

A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus.

Carney complex (CNC) is a familial multiple endocrine neoplasia syndrome associated with GH-producing pituitary tumours and transmitted as an autosomal dominant trait. Mutations of the PRKAR1A gene are responsible for approximately half the known CNC cases but have never found in sporadic pituitary tumours. Pituitary tissue was obtained from an acromegalic CNC patient heterozygote for a common (PRKARIA)i-inactivating mutation. Both immunohistochemistry and electron microscopy showed a highly pleiomorphic pituitary adenoma. The cell culture population appeared morphologically heterogeneous and remained so after more than 30 passages. The mixture was comprised of cells strongly immunostained for GH, spindle-shaped myofibroblast-like cells, and cuboid cells with large axonal projections (negative for GH). The population appeared to have both epithelial and mesenchymal cells. Both at baseline and at passage 30, cytogenetic analysis indicated the presence of normal 46, XY diploid karyotype, whereas losses of the PRKARIA(i) locus were demonstrated in more than 98% of the cells by fluorescent in situ hybridisation, supporting this gene's involvement in pituitary tumorigenesis. Allelic loss may have occurred in a single precursor cell type that differentiated and clonally expanded into several phenotypes. Epithelial-to-mesenchymal transition may also occur in CNC-associated pleiomorphic pituitary adenomas.

Adrenocortical-pituitary hybrid tumor causing Cushing's syndrome.

We describe the first case of an adrenocortical-pituitary hybrid tumor causing Cushing's syndrome in a 17-yr-old boy. Adrenal vein sampling confirmed elevated secretion of both cortisol and ACTH precursors from a right adrenal mass, whereas pituitary ACTH levels, as determined by bilateral inferior petrosal sinus samples (IPSS), were unresponsive to CRH and equal to peripheral levels. There was no biochemical or histological evidence for a pheochromocytoma, but, rather, the tumor demonstrated lipid-rich clear cells characteristic of an adrenocortical adenoma. Immunohistochemical analysis revealed ACTH immunoreactivity and synaptophysin proteins in the tumor. Isolation of tumor cells by the novel technique of laser capture microdissection and subsequent RT-PCR showed expression of POMC messenger ribonucleic acid and cytochrome p450 enzyme messenger ribonucleic acid within the same cells. Finally, ultrastructural analysis provided ultimate proof for adrenocortical-pituitary hybrid cells exhibiting the characteristic vesicular mitochondria and abundant smooth endoplasmic reticulum of steroid cells and the typical secretory granules of corticotrophs within the cytoplasm of the same cells. The adrenocortical tumor expressed the pituitary transcription factor pituitary homeobox factor 1 and the steroidogenic factor 1. The intermingling of the centrally located ectodermally derived pituitary tissue with the mesodermally derived adrenocortical tissue in this adenoma suggests a hitherto unrecognized genetic and phenotypic plasticity within the hypothalamic-pituitary-adrenal axis.

Ceramide mediates nanovesicle shedding and cell death in response to phosphatidylinositol ether lipid analogs and perifosine.

Anticancer phospholipids that inhibit Akt such as the alkylphospholipid perifosine (Per) and phosphatidylinositol ether lipid analogs (PIAs) promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. While investigating the mechanism of Akt inhibition, we found that short-term incubation with these compounds induced rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceramide. Pretreatment with sphingomyelinase inhibitors blocked ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to alkylphospholipids and PIAs in non-small cell lung cancer cell lines. Similarly, exogenous ceramide also decreased active Akt and induced nanovesicle release. Knockdown of neutral sphingomyelinase decreased, whereas overexpression of neutral or acid sphingomyelinase increased cell detachment and death in response to the compounds. When transferred in vitro, PIA or Per-induced nanovesicles increased ceramide levels and death in recipient cells. These results indicate ceramide generation underlies the Akt inhibition and cytotoxicity of this group of agents, and suggests nanovesicle shedding and uptake might potentially propagate their cytotoxicity in vivo.

Pituitary homeobox factor 1, a novel transcription factor in the adrenal regulating steroid 11beta-hydroxylase.

Pituitary homeobox 1 (Ptx1/Pitx1) is a homeodomain-containing transcription factor present throughout pituitary development. Ptx1/Pitx1 interacts with steroidogenic factor 1 (SF-1) in the regulation of pituitary gene expression. SF-1 also plays a critical role in the transcription of enzymes involved in adrenal steroidogenesis. Therefore, we analyzed the presence and role of Ptx1/Pitx1 in human adrenal cortex. Both Ptx1/Pitx1 and SF-1 mRNA were expressed in the human adrenal gland, and immuno-electron microscopy demonstrated the presence of Ptx1/Pitx1 protein in the nucleus of adrenocortical cells. Computer analysis revealed the presence of Ptx1/Pitx1 signal sequences within the promoter region of human 11beta hydroxylase ( hCYP11B1). To examine the role of Ptx1/Pitx1 in the regulation of the genes, we prepared reporter constructs using the 5'-flanking DNA of the hCYP11B1 gene and transfected them into Y-1 mouse adrenocortical cells, HeLa and CV-1 cells. Ptx1/Pitx1 stimulation of hCYP11B1 reporter activity (3-fold over basal) in Y-1 cells was equal to that observed with SF-1. The hCYP11B1 promoter activity in Y-1 cells was not synergistically increased by co-transfection with both Ptx1/Pitx1 and SF-1. Both basal and ACTH-stimulated hCYP11B1 reporter activities in Y-1 cells were increased by co-transfection with either Ptx1/Pitx1 or SF-1 expression vectors. In contrast, co-transfection with both Ptx1/Pitx1 and SF-1 synergistically increased hCYP11B1 promoter activity in HeLa and CV-1 cells (5-fold and 20-fold over basal, respectively). In conclusion, this study represents the first demonstration for a role of Ptx1/Pitx1 in the regulation of transcription of enzymes involved in adrenal steroidogenesis.

Biophysical characterization of gp41 aggregates suggests a model for the molecular mechanism of HIV-associated neurological damage and dementia.

In human immunodeficiency virus (HIV)-infected individuals, the level of the HIV envelope protein gp41 in brain tissue is correlated with neurological damage and dementia. In this paper we show by biochemical methods and electron microscopy that the extracellular ectodomain of purified HIV and simian immunodeficiency virus gp41 (e-gp41) forms a mixture of soluble high molecular weight aggregate and native trimer at physiological pH. The e-gp41 aggregate is shown to be largely alpha-helical and relatively stable to denaturants. The high molecular weight form of e-gp41 is variable in size ranging from 7 to 70 trimers, which associate by interactions at the interior of the aggregate involving the loop that connects the N- and C-terminal helices of the e-gp41 core. The trimers are predominantly arranged with their long axes oriented radially, and the width of the high molecular weight aggregate corresponds to the length of two e-gp41 trimers (approximately 200 A). Using both light and electron microscopy combined with immunohistochemistry we show that HIV gp41 accumulates as an extracellular aggregate in the brains of HIV-infected patients diagnosed with dementia. We postulate that the high molecular weight aggregates of e-gp41 are responsible for HIV-associated neurological damage and dementia, consistent with known mechanisms of encephalopathy.